Use of nerve growth factor for the storage, culture or treatment of cornea

ABSTRACT

The nerve growth factor (NGF) is used for the storage of corneas in culture, for the production and the storage in vitro of single cell populations of the corneal morphological and functional unit (i.e., epithelium, stroma/keratocytes and endothelium) and of the conjunctival epithelium, and for the production and the storage of corneal and conjunctival tissues, in particular for transplantation purposes.  
     The NGF is also proposed for use in the therapy and/or the prophylaxis of diseases of the corneal surface, wherein a lack of integrity of the corneal and conjunctival morphological and functional unit occurs, in particular for pathologies having a dystrophic or neurodystrophic basis, both congenital and acquired.

[0001] The present invention relates to the use of nerve growth factorfor the storage of corneas in culture, the in vitro production ofcorneal and conjunctival tissues and the treatment of corneal andconjunctival diseases. More particularly, the invention concerns the useof the neurotrophin named nerve growth factor (NGF)—known for itsability of influencing the development, the regeneration and themaintenance of the biological functions of neurons—for the treatment ofcorneal and conjunctival tissues, both for properly maintaining theirfunctionality in vitro and for the therapy of pathologies of the ocularsurface.

[0002] As it is known, corneal graft represents the only therapeuticapproach to dystrophic, inflammatory or degenerative corneal diseases inthe event that the said diseases have irreversibly compromised thetransparency or the normal structure of the cornea. Once isolated fromcadaver, the corneas are “stored” up to the time when they aretransplanted, by means of storage methods employing liquids derived fromcell culture mediums. According to one of the most common methods,called “short/intermediate term storage”, the cornea is kept at 4° C. inculture mediums such as McCarey-Kaufman (MK) medium, or in differentcommercial liquids (among which Optisol-GS ), for a maximum period of 7days. A longer term storage is possible with an equally common method,referred to as “organ culture”. This method is based on techniquessimilar to cell culture, and allows to store the explanted corneas, attemperatures comprised between 30 and 36° C., for more than one month.

[0003] None of the above methods affords an improvement of thebiological features of the corneal tissues with the storage, the bestresult achievable being that of maintaining unchanged the quality ofsaid tissues during the storage. This co-operates in making even morecritical the already difficult situation in the field of corneal graft.Actually, more than one half of the corneas are discarded uponexplantation, since the microscope examination shows them to beunsuitable for transplant.

[0004] Another major problem is represented by the need of havingavailable single corneal cell populations, such as endothelial cells,epithelial cells and keratocytes, so as to be able to use them fortransplantation. In particular situations involving the destruction ofthe ocular surface due to physical or chemical burns, or resulting fromsynechiating autoimmune diseases (such as cicatricial pemphigoid,Stevens-Johnson syndrome, etc.) or from post-infectious pathologies, itis of a critical importance to have available corneal epithelial cells(including stem cells) to be transplanted in place of the damaged ocularsurface, and/or to have available conjunctival epithelial tissue for usein replacing the synechiated and retracted surface.

[0005] Considering the pathological situations that may affect themorphological and functional unit consisting of cornea (i.e.,epithelium, stroma/keratocytes and endothelium) and conjunctiva, it isnoted that there is a wide variety of disorders affecting this regionwhich are, to date, of a difficult therapeutic approach, or are totallyorphan of a valid treatment. Such pathologies or situations which mayinterfere with the normal surface integrity include all the impairmentsof the tear film functions, congenital or acquired corneal and/orconjunctival affections such as: neurotrophic and neuroparalytickeratitis and/or conjunctivitis; post-traumatic, post-infectious,post-surgical keratitis and/or conjunctivitis; keratitis and/orconjunctivitis due to laser treatment, to chemical, physical or metalburns; autoimmune, dystrophic, degenerative, post-inflammatorykeratitis. These diseases appear with alterations of the cornealepithelium (spontaneously in the primary neuroparalytic forms or on aphysical, infectious, immune or toxic basis in the secondary forms)which may end in an ulcer, leading in most cases to corneal perforation,either spontaneously or following microtraumas or superinfections. Theclinical picture of this disease is characterised by a slow anddifficult recovery, by superinfections and by the frequent failure ofany therapy. The final outcome is often the opacification of the cornea,or the spontaneous perforation thereof.

[0006] The above diseases are generally treated by having recourse tobandage and to the use of lubricating substances and antibiotics, with aview to prevent the complications. The conjunctival covering isnecessary when the perforation is imminent or has already occurred, andhas the only purpose of preserving the anatomic integrity of theeyeball, while the visual function is sacrificed. The corneal graft,both by lamellar and by penetrating keratoplasty, is absolutelycontra-indicated in view of the frequent occurrence of relapses even onthe transplanted graft, and in view of the risk of superinfections. Onthe basis of in vitro studies the topical medical treatment withpreparations containing fibronectin, plasmin, collagenase inhibitors,EGF (i.e., epidermal growth factor), autoserum has also been proposed.None of these drugs turned out to be resolutive, or able to block or toreduce the corneal injury, or to modify the final outcome of thepathology.

[0007] In particular, in the treatment of corneal burns quite goodresults have been obtained with the transplantation of marginalconjunctiva taken from the contra-lateral eye. When the affection isbilateral, recourse may be had to the donation of conjunctiva by closerelatives or, when this is not possible, to the excision from cadaver.However, this method is not resolutive in the totality of the cases. Inthe case of autograft relapses of the disease may occur after years,while in the case of homograft rejection is frequent, and animmunosuppressive therapy is absolutely necessary, with the well-knownconsequences due to the side-effects.

[0008] Another kind of corneal-conjunctival diseases the treatment ofwhich often gives unsatisfactory results are the herpetic infections.After the resolution of a first pathologic occurrence, herpeticinfections often give rise to relapses, the prevention and the treatmentof which are often unsuccessful.

[0009] Also the diseases affecting the corneal endothelium, such as,e.g., primary and secondary decompensation and endothelitis,characterised by a loss in the number and/or in the function ofendothelial cells, represent a group of affections which presently lackany effective treatment.

[0010] Therefore, it is a primary object of the present invention toprovide a solution to the problems referred to above—which problems areall related to the physiopathology of the corneal and conjunctivalmorphological and functional unit—by means of the use of a therapeuticagent allowing to maintain and restore the proper biological activity ofthe said tissues.

[0011] The molecule known as nerve growth factor (NGF) is the firstcomponent of a complex family of neurotrophins, and is well-known forits trophic, tropic and differentiating action on cholinergic neurons ofthe central nervous system and on the peripheral sympathetic system. NGFis produced in several tissues in mammals, including man, and isreleased in the blood stream at higher levels during the growth and thedifferentiation of the nervous system. Biological, biochemical andmolecular studies carried out in vitro on cell systems have evidenced avery high homology between murine NGF and human NGF. In man as in otheranimal species the NGF is normally present both in the cerebrospinalfluid and in the blood stream at levels of about 10-50 pg/ml. Saidconcentrations increase in some inflammatory pathologies (autoimmunediseases, allergic diseases, etc.) and decrease in other pathologies(diabetes).

[0012] The NGF was discovered in 1951 by Prof. Rita Levi-Montalcini, ofthe Zoology Institute of the Washington University of St. Louis (seeLevi-Montalcini R., Harvey Lect., 60:217, 1966) and represented a majoradvance in the study of the growth and differentiation mechanisms of thenerve cell, as it is capable of influencing the development and themaintenance of the biological functions of neurons and theirregeneration. For the discovery of this molecule, for havingcharacterised its biological role both in the peripheral nervous systemand in the central nervous system, Prof. R. Levi-Montalcini was awardedin 1986 the Nobel prize for Medicine and Physiology.

[0013] Several experimental studies, both in vitro and in vivo, haveshown the physiopathological importance of the NGF in preventingneuronal injury of a surgical, chemical, mechanical and ischemic nature,thereby making it the ideal candidate for use in the therapy of severaldiseases of the central and peripheral nervous system (Hefti F., J.Neurobiol., 25:1418, 1994; J. Fricker, Lancet, 349:480, 1997). Indeed,clinical trials on patients suffering from Parkinson's disease and fromAlzheimer's disease have already started since some years. Said trialsare carried out by intracerebral administration of murine NGF (see,e.g., Olson L. et al., J. Neural Trans.: Parkinson's Disease andDementia Section, 4: 79, 1992). The results of the said studiesconfirmed the observations made on animal models and evidenced theabsence of any possible side-effects upon administration of murine NGF.This feature has more recently been confirmed as regards humanrecombinant NGF (Petty B. G. et al., Annals of Neurology, 36:244-246,1994).

[0014] Ever since its discovery, the studies on the NGF, i.e. on thecharacterisation of its biological, biochemical, molecular, preclinicaland clinical effects have been carried out almost exclusively with NGFisolated from the submaxillary glands of adult rodents; thus, the widestamount of data acquired to date concerns murine NGF. The biochemicalproperties of the latter have been described, in particular, in a workdating back to 1968 (Levi-Montalcini R. and Angeletti P. U.,Physiological Reviews, 48:534, 1968).

[0015] The NGF contained in the salivary glands of mice is a 140 kdaltonmolecular complex, with sedimentation coefficient 7S, consisting ofthree sub-units, α, β e γ, the second one of which represents the trueactive form. This sub-unit, called βNGF, with sedimentation coefficient2.5S, is normally extracted and purified according to three methodswhich are not very different from each other (Bocchini V., Angeletti P.U., Biochemistry, 64: 787-793, 1969; Varon S. et al., Methods inNeurochemistry, 203-229, 1972; Mobley W. C. et al., Molecular BrainResearch, 387: 53-62, 1986).

[0016] The βNGF obtained with such methods is in turn a dimer of about13,000 dalton, consisting of two identical chains of 118 amino acids.Each single chain is stabilised by three disulphide bridges, whilenon-covalent bonds provide to the formation of the dimer structure. Thismolecule, being very stable, may dissolve in almost every solvent, bothaqueous and oily, while keeping unaltered its biochemical features andits biological activity. Further information on the structure and thephysical and biological properties of the molecule can be found inGreene, L. A. and Shooter, E. M., Ann. Rev. Neurosci. 3:353,1980.

[0017] Recently, the structure of the βNGF has been further elucidatedby means of a crystallographic analysis. The latter detected thepresence of three antiparallel couples of strands, with secondarystructure of the β kind, which co-operate in forming a planar surfacealong which the two chains associate to form the active dimer. On saidchains of the βNGF there has been evidenced the presence of four loopregions containing many variable amino acids. To these variable aminoacids is connected, very likely, the specificity of the recognition bythe receptor.

[0018] The biological effect of the NGF is mediated by two receptorspresent on the surface of the respective target cells. There are severalantibodies which selectively inhibit the biological action of NGF. Theexistence of the said antibodies has allowed and allows to accuratelycharacterise and modulate the NGF activity, both in cell systems and invivo.

[0019] In more recent times it has become possible to synthesize humanNGF with genetic engineering techniques (Iwane, M. et al., Biochem.Biophys. Res. Commun., 171:116, 1990), and small amounts of human NGFhave been placed on the market. However, direct experimentation hasshown that the biological activity of human NGF is quite low incomparison with the biological activity of murine NGF. In addition, itis to be considered that almost all of the data presently available onthe effects on man, both in vitro and in vivo, have been obtained byusing murine NGF, and that no adverse effects have been detected whichmay be connected to the murine origin of the product.

[0020] Studies carried out starting from the nineties on animal modelssuggested a possible involvement of the NGF in ocular pathologies.However, such studies almost exclusively concern the use of NGF inretinal affections, i.e. on the nervous tissue, while nothing is presentin the literature about an effect of this neurotrophin on the ocularsurface (i.e., cornea and conjunctiva), nor are there any scientificworks proposing the use of the NGF for the treatment of diseasesaffecting the cornea and/or the conjunctiva. In particular, some studieshave been carried out on animals in order to ascertain the effect of thetopical administration of NGF in the treatment of retinal pathologies,for instance in the treatment of acute retinal ischemy (Siliprandi R. etal., Inv. Ophthalmol. Vis. Sci., 34:3232, 1993), in the transection ofthe optical nerve (Carmignoto G. et al., J. Neurosci., 9:1263, 1989) andin the treatment of retinitis pigmentosa (Lambiase A. e Aloe L.,Graefe's Arch. Clin. Exp. Ophthalmol., 234: S96-S100, 1996). The resultsdemonstrated that the topical administration of NGF can prevent, or atleast delay, the death of the retinal ganglion cells and of thephotoreceptors during the above pathologies. None of these studiesevidenced side-effects on animals.

[0021] On the other hand, with reference to the diseases of the cornealtissues which are the object of interest of the present invention,several Authors have considered for such indications the possibleemployment of the epidermal growth factor (EGF), starting from theassumption that alterations of the corneal epithelium represent thepathogenic key to the development of other corneal affections, such asulcers and keratites.

[0022] The EGF is a 53 amino acid polypeptide discovered andcharacterised in the early sixties (Cohen S., J. Biol. Chem.,237:1555-1562; 1962), having a molecular weight of about 6000 dalton.Said molecule is produced by the salivary gland of adult mice, but ispresent in small amounts in many human tissues. The EGF exerts awell-characterised trophic, proliferative and differentiative action onthe epithelial cells obtained from various animal and human tissues, andits in vitro effectiveness on corneal epithelial cell cultures, as wellas its capacity of inducing proliferation and differentiation of thesecells are described in some experimental works.

[0023] However, the clinical studies on corneal diseases which have beencarried out on the basis of the above experimental data gavecontradictory results, and showed, in general, a substantialineffectiveness of the EGF in this kind of human diseases (Kandrakis A.S. et al., Am. J. Ophthalmol., 98:411, 1984). The said ineffectivenessmay be ascribed, probably, to a different expression of the EGF receptorin vitro and in vivo or, more likely, to the fact that epithelialalterations in corneal diseases would only represent an epiphenomenon ofthe corneal disease itself.

[0024] EP-A-0 312 208 (Ethicon) discloses gel formulations for use inthe treatment of epithelial lesions and epithelial pathologies ingeneral, including lesions and pathologies of the ocular surface. Thesaid formulations contain an active ingredient which may beindiscriminately chosen among the various molecules whose name containsthe expression “growth factor” Although the description is exclusivelyconcerned with the EGF as the preferred active ingredient, and althoughactivity data (in vitro) and formulation examples are given only for theEGF, other growth factors are mentioned as well, such as FGF (fibroblastgrowth factor), PDGF (platelet-derived growth factor), TGF-α(transforming growth factor) or the NGF itself. The said growth factorsare apparently presented as a family of molecules having the samefeatures and the same biological activity as the EGF. As a matter offact, at the current state of the knowledge, the expert is aware thatthe said growth factors have different specific targets and that theyoften have conflicting effects, and does not consider the said growthfactors as biologically equivalent to each other. Besides, as far as theophthalmic field is specifically concerned, the above document describesthe said growth factors as active exclusively on the corneal epithelium,thus reducing the proposed indication to an indication asre-epithelizing factors, useful, in practice, only in traumaticaffections.

[0025] On the contrary, the studies that lead to the present inventionstarted from the concept of cornea as a morphological and functionalunit, wherein the single layers (i.e. epithelium, stroma andendothelium) all carry out a function of an equal importance in thepreservation of the tissues integrity and in the recovery mechanisms. Inthis perspective, a key role is played by the trophic support offered bythe corneal sensory innervation, whose damage brings about a damage tothe whole morphological and functional unit.

[0026] In view of the fact that no evidence existed in the prior art ofan effect of the NGF (i.e., one of the many mediators released by thesensory endings in other regions of the body) on the ocular surface, thequestion has been initially posed whether such neurotrophin might play atrophic-reparatory action on the corneal tissue of neuroectodermalembryonal derivation, since several targets of the said neurotrophinshow a similar embryogenetic derivation. Thus, cornea and conjunctivahave been analysed for the presence of the NGF and of the NGF highaffinity receptor (TrkA, tyrosine kinase A), using a monoclonal antibody(TrkA-antibody; Santa Cruz, USA) with immunohisto-chemistry techniques(Bhattacharyya A. et al., J. Neurosci. 17:7007, 1997). In fact, thepresence of the specific receptor is a key requirement for the activityof the molecule under examination.

[0027] It has thus been found that all of the corneal cells (i.e.,epithelial cells, endothelial cells and keratocytes) express TrkA andthat, at the same time, the corneal somatosensory innervation is able torelease NGF. This discovery allows to hypothesise, on one hand, that theNGF, probably released by the nerve endings, would play aphysiopathologic role in all corneal reparative mechanisms (both at thesurface and at a deeper level) and, on the other hand, that severalsuperficial corneal diseases accompanied by primary damage to theinnervation (such as neurotrophic or dystrophic keratitis andrecurrences of herpetic infections) or with secondary damage to theinnervation (such as chemical or physical burns and post-infectious,autoimmune, post-surgical lesions, or lesions resulting from lasertreatment) would admit as a fundamental etiologic step the lack of NGFrelease.

[0028] The same finding also allows to hypothesise that the NGF would beessential to the maintenance of the whole corneal tissue in culture,i.e. in all such conditions wherein the cornea as a whole, intended as amorphological and functional unit comprising epithelium, stroma andendothelium, or some cells thereof, are deprived of the trophic supportprovided by the release of the NGF by the corneal nerve endings.

[0029] In addition, since the effects that have been observed afteradministration of exogenous NGF occur at concentrations close to thephysiological level it can be hypothesised that a possiblephysiopathogenic mechanism in the corneal affections considered hereinconsists in a reduction of the local levels of the NGF below thethreshold capable of assuring the corneal and/or conjunctival integrity.

[0030] Accordingly, the present invention specifically provides,according to a first aspect thereof, the use of the nerve growth factorfor the storage of corneas in culture and for the storage and theproduction in vitro of corneal and conjunctival tissues, and of singlecorneal or conjunctival cell populations.

[0031] Preferably, the NGF is added to a culture medium of the kindsuitable for the storage of corneas, or for the in vitro culture ofcorneal or conjunctival cells or tissues, in amounts comprised between100 pg/ml and 200 ng/ml, optionally together with other nutrients andother biologically active agents. The NGF to be used to that aim can beof a murine or human origin, including recombinant NGF, and it may beused in lyophilised form, dissolved in solution, in a culture medium orin any other available solvent, so as to obtain a final concentrationcomprised in the range specified above.

[0032] Suitable methods of extracting and purifying the NGF are reportedin the literature mentioned above. For the purpose of experimenting thepresent invention, the Bocchini and Angeletti technique (cited above)has been adopted, the latter being synthetically set forth below. Thesubmaxillary glands of adult male mice are explanted under sterileconditions and the tissues are homogenised, centrifuged and dialysed;then the suspension is passed through subsequent cellulose columns,thereby separating the NGF by adsorption. The NGF is then eluted fromthe column by means of a buffer containing 0.4 M sodium chloride. Thesamples so obtained are analysed by spectrophotometer at a wavelength of280 nm to identify the NGF-containing fractions. The latter aredialysed, and the NGF thus obtained is lyophilised under sterileconditions and kept in refrigerator at −20° C.

[0033] In an evaluation of the effects of an addition of NGF to variousculture mediums for explanted corneas (both at 4° C. and at 30-36° C.),a general improvement of the biological features of the corneal tissuehas been obtained with amounts of NGF comprised between 100 pg/ml and200 ng/ml. The optimal response has been obtained with a concentrationof about 100 ng/ml. Specifically, after 7 days of culture theimprovements obtained were an increase of the endothelial cells density(increase from 10 to 25%), a reduction of the endothelial cellsmortality (absence of trypan blue positive endothelial cells), a betterendothelial morphology (i.e., quantitatively, a 3/3 trophism vs. a 2/3trophism in the controls without addition of NGF), a higher viability ofthe keratocytes and a remarkably better appearance of the epithelium. Inaddition, some corneas that before being placed in culture wereconsidered not suitable for transplantation turned out to be suitableafter being cultured for 7 days in presence of NGF.

[0034] A similar experimental evaluation of the effects of the NGF asadditive in the culture medium of corneal cells (i.e., epithelial cells,endothelial cells and keratocytes) showed that the addition of murineNGF in amounts comprised between 100 pg/ml and 200 ng/ml (with anoptimal response at the concentration of about 100 ng/ml) induces theproliferation and differentiation of the various cell populations, and,moreover, it assists the rooting of the said cell populations on anumber of supporting tissues (such as lamellae of corneal stroma,amniotic membrane, etc.). Further, such addition favours the interactionbetween the different cell types in cell co-cultures. Thus, theinvention may be also employed for culturing mixed corneal cell lines inorder to obtain an artificial cornea useful for transplantation.

[0035] Also the conjunctival epithelial cell cultures have shown aproliferation and differentiation increase, as well as an increase inthe number of goblet cells, when cultured in presence of murine NGF atthe same concentrations specified above. The graft of epithelialconjunctival tissue thus obtained is extremely advantageous forreplacing the conjunctiva of patients suffering fromkerato-conjunctivitis sicca (dry eye syndrome), both primary andsecondary to other pathologies.

[0036] According to another aspect of the invention, there is provided,therefore, a culture medium for the storage of corneas or for thestorage and the production in vitro of corneal and/or conjunctivaltissues, or of single corneal or conjunctival cell populations,characterised in that it contains an effective amount of NGF, preferablycomprised between 100 pg/ml and 200 ng/ml, most preferably about 100ng/ml.

[0037] As set forth before, according to a further aspect of theinvention there is provided the use of the nerve growth factor in themanufacture of a medicament for the therapy and/or the prophylaxis ofcorneal and/or conjunctival diseases.

[0038] Specifically, the invention is useful for the production ofmedicinal products suitable for the treatment and/or the prophylaxis ofcongenital and/or acquired corneal and/or conjunctival diseases chosenfrom the group consisting of: neurotrophic and neuroparalytic keratitisand/or conjunctivitis; herpetic keratitis and/or conjunctivitis;post-traumatic, post-infectious, post-surgical keratitis and/orconjunctivitis; keratitis and/or conjunctivitis due to impairment of thetear film functions, to laser treatment, to chemical, physical or metalbums; autoimmune, dystrophic, degenerative and post-inflammatorykeratitis.

[0039] Preferably, a medicinal product according to the inventionsuitable for topical administration contains, alone or in combinationwith one or more other active ingredients, from 10 to 500 μg of NGF perml, an optimal concentration being about 250 μg of NGF per ml. Suchproduct may be in the form of ophthalmic solution for eye-drops, or inthe form of a gel, an ointment, a cream or a powder, or it can be addedto a local bandage or to a medicinal contact lens.

[0040] According to another embodiment of the invention, thepharmaceutical product for the manufacture of which the NGF is proposedis a medicament indicated for the therapy and/or the prophylaxis ofprimary and secondary endothelial ophthalmic pathologies. For this use,the preferred formulations contain (also in this case optionally incombination with other active ingredients) from 1 to 250 μg of NGF perml, and the administration is carried out by introduction in theanterior chamber of the eye.

[0041] In vivo studies on animals concerning the treatment of disordersof the corneal endothelium have been carried out by administration inthe anterior chamber of the eye of aqueous solutions containing NGF atconcentrations comprised between 1 and 250 μg/ml. In particular, inrabbits wherein an endothelial damage had been introduced by means of acryoprobe a complete restoration of the endothelial density has beenobtained after 15 days of treatment with NGF. The administration of NGFin endothelial pathologies, both of a dystrophic nature and acquired,both with loss of the number of endothelial cells and with loss of thefunctionality thereof, has been shown to restore a proper endothelialfunction.

[0042] In a series of studies on man for ascertaining the efficiency ofthe treatment with NGF in the topical treatment of corneal and/orconjunctival affections, a number of pathologies have been selected onthe basis of a primary or secondary involvement of the somato-sensorialcorneal nerve plexus. Murine NGF (2.5S), obtained with the purificationmethod summarised above, has been employed in such studies. The NGF hasbeen topically administered at a concentration of about 250 mg/ml,diluted in balanced salt solution.

[0043] The table shown further on summarises the results obtained from astudy on 5 patients affected by torpid corneal ulcer due toneurodistrophic keratitis (2 eyes), or after keratoplasty (2 eyes) ordue to bum by alkali (2 eyes). The therapeutic schedule consisted in theinstillation of one-two drops of the preparation of the invention with adaily frequency divided as follows: every 2 hours for the first 2 days,6 times a day up to the 2nd day after the complete re-epithelization ofthe cornea and twice a day for the following 15 days.

[0044] The topical treatment with the NGF was started after 15 days oftreatment with autoserum, in the absence of signs of improvement of theclinical picture. All of the patients treated showed clear signs ofrecovery within 2 weeks from the start of the treatment with NGF, andnone of them evidenced any occurrence of local or systemic side-effectsduring the treatment or in the following period. Once suspended, thetherapy should be immediately restored in the event that the first signsor symptoms of any relapse of the epithelial pathology appear.

[0045] The concerned 5 cases are discussed in detail below, and the maindata are summarised in the following table.

[0046] 1 st Case

[0047] A 9-year-old female, affected by a congenital anophthalmos,showed in the other eye a corneal ulcer since more than 20 days. Thesaid ulcer, topically treated with antibiotics and steroids, did notshow any tendency to healing. The ulcer was about 7 mm in diameter andmore than ⅔ of the corneal stroma in depth. The clinical examinationshowed corneal anaesthesia, on the basis of which a neurotrophickeratitis was diagnosed. After 15 days of treatment with autoserum,having ascertained a progressive worsening of the clinical picture, thetopical treatment with NGF was started.

[0048] After 4 days the corneal ulcer was reduced to about a 3.5 mm indiameter, and after 12 days the cornea was completely healed and thetreatment with the NGF was discontinued. The patient showed a centralleukoma with some neo-vessels and, although reduced, a cornealsensitivity was present, while it was completely absent before thetreatment. After a 8 months follow-up the patient showed {fraction(5/10)} of visual acuity and the corneal sensitivity was still present.

[0049] 2nd Case

[0050] A 26-years-old woman, affected by syndactilia and deficit of theVIII pair of cranial nerves, showed since about 2 months a corneal ulcerwhich progressively deepened until it reached an extent of 7 mm and adepth up to the Descemet membrane. The clinical examination showed acomplete corneal anaesthesia.

[0051] After 2 weeks of therapy with the NGF apparent signs of recoverywere present, with a reduction in the ulcer depth. After 6 weeks oftherapy the cornea was completely re-epithelized, while a centralleukoma with some neo-vessels was left. In addition, some cornealsensitivity was recovered, although reduced.

[0052] 3rd Case

[0053] A man aged 25, who had undergone enucleation of the left eyefollowing the onset of a corneal ulcer after a penetrating keratoplasty,showed since about 1 month a corneal ulcer in the right eye, into acorneal graft. The corneal ulcer was about 5 mm in diameter and itsdepth was ½ of the corneal stroma. Corneal anaesthesia was also present.

[0054] After 2 weeks of treatment with NGF a corneal healing process wasevident, with reduction of the extent and the depth of the ulcer. After4 weeks of treatment with NGF the ulcer was completely healed and acentral leukoma with some neo-vessels was left. A marked cornealhypoesthesia was still present.

[0055] 4th Case

[0056] A 56-years-old man showed a bilateral corneal ulcer due to a burnby alkali. The right eye, after having undergone surgery by penetratingkeratoplasty, had been enucleated after the onset of an ulcer. The lefteye, after undergoing lamellar keratoplasty, developed a torpid ulcer ofabout 7 mm in diameter, which did not show any sign of healing.

[0057] After 2 weeks of treatment with NGF the onset of a recoveryprocess was evident, and the said process was complete after 5 weeks.

[0058] 5th Case

[0059] A 56-years-old man showed a bilateral corneal ulcer due to a burnby hydrochloric acid. In the right eye an ulcer of about 4 mm indiameter was present, while in the left eye the ulcer was bigger (morethan 8 mm in diameter) and deeper. The patient showed a marked cornealhypohestesia.

[0060] After 2 weeks of treatment with NGF the right eye was completelyhealed, with a residual central leukoma, while the ulcer in the left eyewas reduced both in extent and in depth, although a neo-vessel pannuswas present. After 3 weeks of treatment both corneas were fullyre-epithelized, a central leukoma was left and corneal sensitivity waspresent, although reduced. After a 2 months follow-up both corneasremained re-epithelized, and the visual acuity of the right eye was{fraction (3/10)}, while that of the left eye was {fraction (1/10)}. Inaddition, a corneal sensitivity was still present in both eyes. TABLE 1Treatment in vivo with Nerve Growth Factor Age Time Treat- Patient(years), from Initial Previous ment Final sensi- Follow No. Pathologysex onset sensitivity therapy with NGF Outcome tivity Visus up 1neurotrophic  9, F 20 days anaesthesia antibiotics, 12 days recoverynormal/ improved 8 months ulcer steroids hypoesthesia autoserum 2neurotrophic 26, F 2 months anaesthesia antibiotics, 6 weeks recoverynormal/ improved 4 months ulcer steroids, hypoesthesia autoserum 3neurotrophic 25, M 1 month anaesthesia antibiotics, 4 weeks recoveryanaesthesia improved 2 months ulcer in PK^(a)) autoserum 4 post(burn by56, M 2 months anaesthesia antibiotics, 5 weeks recovery normal/improved 4 months alkali) ulcer autoserum hypoesthesia in LK^(b)) 5 (RE)post(burn by 56, M 1 month anaesthesia/ antibiotics, 2 weeks recoverynormal/ improved 3 months acid) ulcer hypoesthesia autoserumhypoesthesia 5 (LE) post(burn by 56, M 1 month anaesthesia/ antibiotics,3 weeks recovery normal/ improved 3 months acid) ulcer hypoesthesiaautoserum hypoesthesia

[0061] The foregoing data clearly show the effectiveness of the use ofthe NGF not only for storing and producing in vitro corneal and/orconjunctival tissue, as a whole or partially, or for storing andproducing in vitro the single cell types of which the said tissuesconsist, but also for treating and preventing human or animal diseasesaffecting the corneal morphological and functional unit or theconjunctiva, which diseases have not found, so far, an effectivetherapy.

[0062] The present invention has been disclosed with particularreference to some specific embodiments thereof, but it should beunderstood that modifications and changes may be made by the personsskilled in the art without departing from the scope of the invention asdefined in the appended claims.

1. Use of the nerve growth factor (NGF) for the storage of corneas inculture and for the storage and the production in vitro of corneal andconjunctival tissues, and of single corneal or conjunctival cellpopulations.
 2. Use according to claim 1, wherein the NGF is added to aculture medium for the storage of corneas, or to a culture medium forthe storage and the production of corneal or conjunctival cells ortissues, at a concentration between 100 pg/ml and 200 ng/ml.
 3. Useaccording to claim 2, wherein the said concentration is about 100 ng/ml.4. Use according to any one of the preceding claims, wherein the saidculture medium also contains additional nutrients and/or biologicallyactive agents.
 5. Use of the nerve growth factor (NGF) in themanufacture of a medicament for the therapy and/or the prophylaxis ofcorneal and/or conjunctival diseases.
 6. Use according to claim 5,wherein the said medicament is indicated for the treatment and/or theprophylaxis of congenital and/or acquired corneal and/or conjunctivaldiseases chosen from the group consisting of: neurotrophic andneuroparalytic keratitis and/or conjunctivitis; herpetic keratitisand/or conjunctivitis; post-traumatic, post-infectious, post-surgicalkeratitis and/or conjunctivitis; keratitis and/or conjunctivitis due toimpairment of the tear film functions, to laser treatment, to chemical,physical or metal burns; autoimmune, dystrophic, degenerative andpost-inflammatory keratitis.
 7. Use according to claims 5 or 6, whereinthe said medicament is for topical administration, and contains from 10to 500 μg of NGF per ml.
 8. Use according to claim 7, wherein saidmedicament contains about 250 μg of NGF per ml.
 9. Use according toclaim 5 wherein the said medicament is indicated for the therapy and/orthe prophylaxis of primary and secondary endothelial ophthalmicpathologies.
 10. Use according to claim 9, wherein the said medicamentis for administration by introduction in the anterior chamber of theeye, and contains from 1 to 250 μg of NGF per ml.
 11. Use according toany one of claims 5-10, wherein the NGF is in combination, in the saidmedicament, with one or more other active ingredients.
 12. Use andculture medium according to any one of the previous claims, wherein thesaid NGF is of murine or of human origin, or it is human recombinantNGF.